It has recently been discovered that prokaryotes can acquire resistance to viruses and plasmids by integrating short fragments of foreign DNA into clusters of regularly interspaced short palindromic repeats (CRISPR's). These repeats are then transcribed and processed into small guide RNA's that are used to direct the destruction of foreign nucleic acid. This mechanism has many parallels with eukaryotic RNA interference but the proteins that are associated with the CRISPR response are evolutionarily unrelated to their eukaryotic counterparts. Our long-term goal is to understand the biochemical and structural basis of CRISPR-mediated resistance in prokaryotes. The objective here is to determine the mechanisms used to produce guide RNA's from CRISPR transcripts. Despite recent advances, understanding of these mechanisms is rudimentary. Our objective will be achieved through biochemical, structural and cell based analyses of CRISPR transcripts and the CRISPR-associated (cas) proteins. We hypothesize that in all prokaryotes this process will require the specific and sequential action of multiple cas proteins and that the fundamental mechanism will be conserved. Successful completion of the proposed studies is significant because it will increase our understanding of bacterial resistance to viruses and plasmids. Both of these genetic elements play important roles in the genetics of pathogenic bacteria. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because it will increase our understanding of the interplay between pathogenic bacteria and mobile genetic elements, such as viruses and plasmids. This interplay is instrumental to the acquisition of antibiotic resistance, and evolution, of pathogen bacteria. Thus, the proposed research is relevant to the NIH's goals of improving the control of disease, enhancing human health and advancing our understanding of biological systems.